Miniature support for thin films containing single channels or nanopores and methods for using same

ABSTRACT

Single-channel thin film devices and methods for using the same are provided. The subject devices comprise cis and trans chambers connected by an electrical communication means. At the cis end of the electrical communication means is a horizontal conical aperture sealed with a thin film that includes a single nanopore or channel. The devices further include a means for applying an electric field between the cis and trans chambers. The subject devices find use in applications in which the ionic current through a nanopore or channel is monitored where such applications include the characterization of naturally occurring ion channels, the characterization of polymeric compounds, and the like.

CROSS REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e), this application claims priority to thefiling date of the United States Provisional Patent Application Ser. No.60/107,307 filed Nov. 6, 1998, the disclosure of which is hereinincorporated by reference.

ACKNOWLEDGMENT

This invention was made with Government support under Grant No. HG01360awarded by the National Institutes of Health. The United StatesGovernment has certain rights in this invention.

INTRODUCTION

1. Field of the Invention

The field of this invention is ion channels or nanopores, particularlymethods of measuring the ionic current flowing through ion channels ornanopores.

2. Background of the Invention

Methods of measuring the ionic current through a single ion channel arecritical to the study of ion channels, which play pivotal roles in avariety of physiological processes. Through such methods, the processesunderlying ion permeation and gating have been explored.

One approach for measuring the ionic current flowing through a singleion channel is the patch-clamp technique. In the patch-clamp technique,a small patch of membrane that includes an ion channel of interest isisolated at the tip of a glass micro-electrode. The ion current flowingthrough the isolated ion channel is then measured. This approach hasbeen invaluable as a research tool, but suffers from limitations incertain circumstances. For example, not all ion channels of interest areaccessible by patch-clamp techniques. In addition, patch-clamptechniques do not provide the ability to modulate the membrane componentand thus explore the lipid/channel interactions that potentially affectcurrent flow through the channel.

In an alternative approach that can overcome these limitations, thechannel of interest is reconstituted in an artificial thin film device.Although several such devices have been developed since the 1960s, thereis continued interest in new configurations that reduce capacitance,noise, and solution volume.

Relevant Literature

Of interest are Wonderlin et al., “Optimizing planar lipid bilayersingle-channel recordings for high resolution with rapid voltage steps”Biophys. J. (1990) 58:289-297; Brutyan et al., “Horizontal‘solvent-free’ lipid bimolecular membranes with two-sided access can beformed and facilitate ion channel reconstitution,” Biochimica etBiophysica Acta, (1995) 1236: 339-344; and Kasianowicz, et al.,“Characterization of individual polynucleotide molecules using amembrane channel,” Proc. Natl. Acad. Sci. USA (1996) 93: 13770-13773.

SUMMARY OF THE INVENTION

Miniature thin film support devices and methods for using the same areprovided. In the subject devices, an electrical communication means,e.g. a U tube, connects cis and trans chambers that are filled with anaqueous fluid. At the cis end of the electrical communication means is aconical aperture that is sealed with a thin film into which has beeninserted a single nanopore or channel. The subject devices furtherinclude a means for applying an electric field between the cis and transchambers. The subject devices find use in a variety of applications inwhich the ionic current through the inserted nanopore or channel ismonitored or measured for a period of time, e.g. several hours,including the characterization of naturally occurring ion channels, thecharacterization of polymeric compounds, and the like.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Horizontal thin film apparatus according to the subjectinvention. A 0.8 cm inside diameter U-shaped tube connects two 65 μlbaths (A & B) milled into a Teflon support. The baths and the Teflontube are filled with 1 M KCl buffer. The chamber is connected to anAxopatch 200B amplifier via AgCl electrodes that are pressure fittedinto the sides of the two baths. One end of the Teflon U tube has aconical tip that narrows abruptly to a 25 μm conical aperture.Diphytanoyl PC/hexadecane bilayers are formed across this aperture.α-toxin is inserted into the bilayer following addition of 0.04 μg tobath A. Nucleic acids are driven through the toxin channel by an appliedvoltage of 120 mV (+at the trans side (bath B)).

FIG. 2. Typical blockades of monovalent ion current in the α-hemolysinpore caused by A(30)C(70)Gp RNA observed with the device shown inFIG. 1. A single α-hemolysin channel was inserted into the bilayer withan open current of 126 pA at 120 mV in 1 M KCl buffer. Following controlmeasurements in the absence of RNA, A(30)C(70)Gp RNA was added to theciv bath at 100 μg mL⁻¹. Each event represents translocation of a singleRNA molecule across the pore. In this experiment, most biphasic eventshad the orientation 5 pA residual current (95 percent current blockade)first followed by a 19 pA residual current 84 percent blockade). Thiscorresponds to the polyC segment at the 3′ end of the molecule enteringthe pore first. The opposite orientation (insert) constituted less than10% of the blockade events. The solid arrows highlight monophasiccurrent blockades of approximately 95% and 84%; the dashed arrowhighlights a permablock that required a voltage reversal to be cleared.This experiment is representative of four experiments with A(30)C(70)GpRNA in which successful preparative cutting of the T7 RNA polymeraseproduct by T1 RNase was confirmed by PAGE.

FIG. 3. Effect of ribonuclease A addition upon the frequency of biphasicblockades caused by A(30)C(70)Gp RNA observed in the device of FIG. 1. Asingle α-hemolysin channel was inserted into the bilayer with an opencurrent of 125 pA at 120 mV in 1 M KCl buffer at room temperature.Following control measurements in the absence of RNA, A(30)C(70)Gp RNAwas added to the cis bath for a final concentration of 100 μg mL⁻1.Channel blockades were recorded for ten minutes, and then ribonuclease A(20 μg mL⁻1) was added to the cis bath. Blockades were then acquired intwo minute increments at intervals up to 20 minutes. a) Frequency of95%-to-84% biphasic blocks similar to those shown in FIG. 2. b)Frequency of monophasic blockades that reduce the channel current by 95%as observed in experiments with polyC alone. c) Frequency of monophasicblockades that reduce the channel current by 84 percent as observed withpolyA alone. In all cases, events were counted if the specified blockadeamplitude was achieved for a minimum of 50 μs.

DETAILED DESCRIPTION OF THE INVENTION

Single-channel thin film devices and methods for their use are provided.The subject devices have a cis chamber connected to a trans chamber viaan electrical communication means, e.g. a U-shaped conductor. At the cisend of the electrical communication means is a conical aperture sealedwith a thin film having a single nanopore or channel. The subjectdevices further include a means for applying an electric field betweenthe cis and trans chambers, e.g. cis and trans electrodes. The subjectdevices find use in a variety of different applications in which theionic current through a nanopore or channel is monitored. In furtherdescribing the subject invention, the subject devices will be describedfirst followed by a review of a number of different representativemethods in which the subject devices find use.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

The Subject Devices

As summarized above, the subject single-channel thin film devicesinclude the following elements: (a) a cis chamber; (b) a trans chamber;(c) an electrical communication means connecting the cis and transchambers; and (d) a thin film at the cis terminus of the electricalcommunication means that contains a single nanopore or channel.

The cis and trans chambers may have any convenient configuration. Assuch, the cis and trans chambers may have a conical, cylindrical, cube,or other shape as desired. The volume of the chambers may vary as well,where the volume of each chamber is at least about 1 μl, usually atleast about 10 μl and more usually at least about 50 μl, and may be aslarge as 1 ml or larger, but will usually not exceed about 2 ml and moreusually will not exceed about 10 ml. In certain preferred embodiments,e.g. where microgram quantities of nucleic acid are analyzed, asdescribed in greater detail below, the chambers will have relativelysmall volumes, ranging from about 1 μl to 10 μl and usually from about10 μl to 50 μl. The shape and volume of the cis and trans chambers maybe the same or different, such that the shape or volume of the cischamber may be substantially similar to that of the trans chamber ordifferent from that of the trans chamber.

Connecting the cis and trans chambers is an electrical communicationmeans. By electrical communications means is meant a conduit or vesselthat is capable of holding a conductor through which an electricalcurrent can flow, e.g. an electrolyte solution. In a typicalapplication, the conduit or vessel has an opening in the cis chamber andthe trans chamber, i.e. it has an open cis end and an open trans end,thereby allowing for fluid flow and, importantly, ionic current flowunder appropriate conditions, e.g an applied electric field. The conduitor vessel may have a variety of different cross-sectional shapes, wherevarious cross-sectional shapes of interest include circular, square,oval, rectangular, trapezoidal, and the like. In general, the averagecross-sectional area along the entire electrical communication meanswill be at least about 10 μm², usually at least about 50 μm² and moreusually at least about 500 μm², where the cross-sectional area may be aslarge as 2mm² or larger, but will usually not exceed about 1 mm² andmore usually will not exceed about 0.6 mm². In preferred embodiments,the electrical communication means is a tubular structure that has acircular cross-sectional shape along its entire length. In thesepreferred embodiments, the average diameter along the entire length ofthe electrical communication means is at least about 10 μm, usually atleast about 50 μm and more usually at least about 500 μm, where thediameter may be a large as 2 mm or larger, but will generally not exceedabout 1 mm and usually will not exceed about 0.8 mm. At least the cisend of the electrical communication means enters the cis chamber throughthe floor or wall of the cis chamber. The cis end may be flush with thefloor or wall of the cis chamber or extend a small distance into the cischamber, where that distance will not exceed about 2 mm and usually willnot exceed about 1 mm. In many embodiments, the trans end will beassociated with the trans chamber in an analogous fashion. In suchembodiments, the electrical communication means generally is the shapeof a “U,” e.g. where the electrical communication means is a U-shapedpatch tube filled with an electrolyte solution. The length of theelectrical communication means typically ranges from about 0.5 mm to 5mm, usually from about 1 mm to 4 mm and more usually from about 2 mm to3 mm.

At the cis end of the electrical communication means is a conicalaperture (or opening) of μm dimensions, e.g. a conical fitting or capwith a μm sized opening. In other words, the cis end of the electricalcommunication means has an internal conical bore with a hole at the end.As the aperture or opening is of μm dimensions, it typically has adiameter ranging from about 1 to 100 μm, usually from about 5 to 50 μmand more usually from about 10 to 25 μm. The cis end of the electricalcommunication means may be fabricated such that it gradually narrows atthe cis end to provide for a conical aperture of μm dimensions (i.e. theconical aperture may be part of the electrical communication means), orthe cis end may be capped with a separate conical aperture component orelement that fits over or caps the cis end or terminus. In a preferredembodiment, the opening of the conical aperture at the cis end ishorizontal, i.e. it is parallel to the water line of fluid, whenpresent, in the cis chamber and the horizon of the substrate on whichthe device rests.

The horizontal aperture at the cis end of the electrical communicationmeans is sealed with a thin film, such as a lipid bilayer. A variety ofdifferent lipid bilayers are known in the art and may be used to producethe thin film and seal the horizontal cis conical aperture.Representative lipid bilayers included those prepared from one or morelipids of the following group: phosphatidlycholine, phosphatidylserine,phosphatidylethanolamine, glycerol mono-oleate, cholesterol, etc. Thethin film may also be formed by inorganic materials such as siliconnitride, and the like.

Inserted into the horizontal bilayer is a single channel or nanoporethrough which ionic current can flow, e.g. from the cis to the transside of the pore upon application of an applied electric field. As usedherein, the terms “nanopore” and “channel” are used interachangeably torefer to structures having a nanoscale passageway through which ioniccurrent can flow. The inner diameter of the nanopore may varyconsiderably depending on the intended use of the device. Typically, thechannel or nanopore will have an inner diameter of at least about 0.5nm, usually at least about 1 nm and more usually at least about 1.5 nm,where the diameter may be as great as 50 nm or longer, but in manyembodiments will not exceed about 10 nm, and usually will not exceedabout 2 nm. In those preferred embodiments in which the subject deviceis designed to characterize polymeric molecules as described incopending application Ser. No. 08/405,735 entitled “Characterization ofIndividual Polymeric Molecules Based on Monomer Interface Interactions,”(UC Reference No. 91-287-2) the inner diameter of the nanopore may besufficient to allow translocation of singled stranded, but not doublestranded, nucleic acids. As such, in these preferred embodiments, theinner-diameter will be at least about 1 nm, usually at least about 1.5nm and more usually at least about 2 nm, but will not exceed about 3 nm,and more usually will not exceed about 5 nm.

The nanopore should allow a sufficiently large ionic current under anapplied electric field to provide for adequate measurement of currentfluctuations. As such, under an applied electric field of 120 mV in thepresence of pH 7.5 buffered solution (as described in the experimentalsection, infra), the open (i.e. unobstructed) nanopore should providefor an ionic current that is at least about 1 pA, usually at least about10 pA and more usually at least about 100 pA. Typically, the ioniccurrent under these conditions will not exceed about 0.5 nA and moreusually will not exceed about 1 nA. In addition, the channel shouldprovide for a stable ionic current over a relatively long period oftime. Generally, channels finding use in the subject devices provide foraccurate measurement of ionic current for at least about 1 min, usuallyat least about 10 minand more usually at least about 1 hour, where theymay provide for a stable current for as long as 24 hours or longer.

The single nanopore that is inserted into the lipid bilayer may be anaturally occurring or synthetic nanopore. Typically the nanopore willbe a proteinaceous material, by which is meant that it is made up of oneor more, usually a plurality, of different proteins associated with eachother to produce a channel having an inner diameter of appropriatedimensions, as described above. Suitable channels or nanopores includeporins, gramicidins, and synthetic peptides. Of particular interest isthe heptameric nanopore or channel produced from α-hemolysin,particularly αhemolysin from Staphylococcus aureus, where the channel ispreferably rectified, by which is meant that the amplitude of thecurrent flowing in one direction through the channel exceeds theamplitude of the current flowing through the channel in the oppositedirection.

The single-channel thin films of the device are configured so as toprovide for high resistance, low noise and stability. As such, theresistance of the subject single-channel bilayers is at least about 1gigaohm, usually at least about 10 gigaohm and more usually at leastabout 200 gigaohm, where the resistance may be as high as 500 gigaohm orhigher. The noise preferably does not exceed about 0.6 pA and usuallydoes not exceed about 0.5 pA RMS at 5 kHz bandwidth in whole cell mode,and does not exceed about 0.4 pA and usually does not exceed about 0.2pA RMS in patch mode. Furthermore, the subject single channel bilayersare stable for period of at least about 1 min, usually at least about 1hour under an applied electric field of 100 mV or more, where thesubject bilayers may be stable for much longer periods under the sameconditions, e.g. they may be stable for periods of 24 hours or longer.In addition, the capacitance of the bilayer ranges from about 0.3 to 1.5μF cm⁻², usually from about 0.4 to 1.2 μF cm⁻² and more usually fromabout 0.3 to 0.4 μF cm⁻².

The subject devices also generally comprise a means for applying anelectric field between the cis and trans chambers, and therefore betweenthe cis and trans sides of the bilayer and single nanopore presenttherein. The electric field applying means is typically capable ofgenerating a voltage of at least about 10 mV, usually at least about 50mV and more usually at least about 100 mV. Typically, the electric fieldgenerating means is made up of silver chloride electrodes positioned inthe cis and trans chambers that are connected to a voltage source.

The device typically further comprises a means for monitoring thecurrent flow through the channel and processing the observed currentflow to produce a usable output. Generally, such monitoring meansincludes a very low noise amplifier and current injector, and an analogto digital (A/D) converter. The device may further comprise otherelements of the output generating system, including data acquisitionsoftware, an electronic storage medium, etc. A suitable system isdescribed in the experimental section, infra.

The cis and trans chambers may be fabricated from a wide variety ofmaterials. Typically these components will be fabricated or at leastlined with a relatively inert material, such as a polymeric material,e.g. Teflon. The components may be fabricated using any convenienttechnique, e.g. machining.

Preparation of the Subject Devices

The subject devices may be prepared as follows. The cone-shaped bore inthe electrical communication means (U tube) is most easily produced bymolding heat shrinkable Teflon tubing (Cole-Parmer) around a steelmandril that has been machined into the appropriate shape (e.g. amandril prepared from an 0.80 mm stainless steel straight rod, with atip ground to a highly polished 80° point, where the tip is notrounded). After removing the mandril, a microtome is used to cut awayexcess Teflon from the tip until a hole of the desired size is produced.A typical hole is in the range of 20-40 micrometers. The subject processis further disclosed in FIG. 6 of priority application Ser. No.60/107,307, the disclosure of which is herein incorporated by reference.The U-tube or electrical communication means is then threaded into aTeflon holder thus connecting the cis and trans chambers, such that thecis end of the electrical communication means is horizontal, e.g. arisesfrom the floor of the cis chamber. In a preferred embodiment, the cisand trans chambers, electrical communication means and conical apertureare assembled to produce a device as shown in FIG. 1. In FIG. 1, device10 comprises cis chamber or bath 1 and trans chamber or bath 2.Connecting the floors of cis and trans chambers is patch tube 3. At thecis end of the electrical communication means is conical aperturecapping element 4 comprising aperture 5 (e.g. 25 μm aperture). Alsopresent are electrodes 6 and 7.

Following assembly of the above components, the cis and trans chambersmay be cleaned as desired. See the experimental section, infra, for aspecific representative cleaning protocol. Following cleaning, theaperture is then typically coated with a lipid solution dissolved in asuitable solvent, typically an organic solvent, where the solvent isthen evaporated from the aperture to leave a dry, lipid coated aperture.Next, the cis and trans chambers, as well as the electricalcommunication means, are filled with an appropriate buffered medium,e.g. a buffered salt solution (such as a 1.0 M KCl solution) at pHranging from about 5 to 9, usually from about 7 to 8. Electrodes capableof serving as the applied electric field generating means are thenplaced into the trans and cis chambers. See FIG. 1 in which theelectrodes are indicated as elements 6 and 7.

The next step in the fabrication process is to seal the aperture with athin film. One protocol for sealing the aperture with a lipid bilayer isto paint the lipid bilayer onto the aperture. In painting the lipidbilayer onto the aperture, a bristle of sufficient dimensions, e.g. 10to 200 μm diameter, usually 50 to 100 μm diameter, is dipped into asuitable lipid solution (e.g. lipid in organic solvent, concentrationrange from about 1 to 5 mg per ml, usually from about 2 to 4 mg per ml).The dipped bristle is then gently brushed against the aperture, whichresults in the formation of a lipid bilayer that seals the aperture. Theseal is then tested and the aperture may be brushed repeatedly with aclean bristle until a bilayer with the desired capacitance is obtained.

The final step in the preparation of the subject device is the insertionof the nanopore into the lipid bilayer. Typically, an aqueous nanoporeor channel comprising solution is introduced into the cis chamber and anelectric field is applied across the lipid bilayer in a mannersufficient for a single channel to insert or intercalate into the lipidbilayer. The nanopore or channel concentration in the cis bath followingintroduction of the stock solution (i.e. the solution comprising thenanopore or channel) ranges from about 0.8 ug per ml to 5 ug per ml,usually from about 1 ug per ml to 4 ug per ml and more usually fromabout 1.2 to 2.5 ug per ml. The voltage applied between the cis andtrans sides of the bilayer ranges from about 10 to 200 mV, usually fromabout 100 to 150 mV.

Following insertion of a single nanopore into the bilayer, the device isready for use in applications where ionic current through the singlechannel is monitored.

Uses of the Subject Devices

The subject devices find use in a variety of different applications inwhich the ionic current through a nanopore or channel is monitored.Representative applications in which the subject devices find useinclude: (a) the study and characterization or analysis of naturallyoccurring ion channels or ion permeable passages; and (b) thecharacterization of polymeric compounds, e.g. the determining of thebase sequence of a nucleic acid; and the like.

Where the device is used to characterize the properties of a naturallyoccurring ion channel, the nanopore that is inserted or present in thelipid bilayer covering the aperture is the ion channel of interest. Theionic current through the ion channel is then measured under variousconditions, e.g. in the presence of various buffer solutions, agents,lipid bilayers and the like, so as to characterize the ion channel. Forexamples, see Wonderlin et al., “Optimizing planar lipid bilayersingle-channel recordings for high resolution with rapid voltage steps”Biophys. J. (1990) 58:289-297; and Brutyan et al., “Horizontal‘solvent-free’ lipid bimolecular membranes with two-sided access can beformed and facilitate ion channel reconstitution,” Biochimica etBiophysica Acta, (1995) 1236: 339-344.

The subject devices also find use in methods of characterizing polymericmolecules, e.g. determining the sequence of bases in a given nucleicacid. In such methods, the polymer is moved relative to the nanopore ina manner such that each different monomeric unit of the polymer causes acorrespondingly different current to flow through the nanopore. Forexample, a single stranded nucleic acid may be translocated through thenanopore and the effect of each base on the current flowing through thenanopore monitored and recorded. From the resultant recorded currentfluctuations, the base sequence of the nucleic acid can be determined.Methods of characterizing polymeric molecules in this manner are furtherdescribed in application Ser. No. 08/405,735 and entitledCharacterization of Individual Polymer Molecules Based onMonomer-interface Interactions (UC Ref: 91-287-2), the disclosure ofwhich is herein incorporated by reference.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

I. Preparation of a Horizontal Bilayer Containing a Single Channel Usinga Miniature Horizontal Support

A single channel was inserted into a bilayer on the horizontal apertureas follows.

A. Formation of diphytatnoyl PC/hexadecane bilayers on a horizontalaperture.

A miniature support was manufactured as described under ‘Preparation ofSubject Devices’, supra. A lipid bilayer was then formed as follows: Theaperture and the Teflon bath holding the aperture were first cleaned for10 min in boiling 5% nitric acid, then rinsed in nanopure water. Justbefore use, the aperture and bath were rinsed with ethanol followed byhexane, and then air dried. The aperture was then coated with a thinfilm of diphytanoyl PC (obtained from Avanti Polar Lipids, Birmingham,Ala.) by applying 5 μl of a 200 μg per mL solution in spectroscopy gradehexane which was then evaporated with a light stream of air injectedthrough the U-tube from the trans side. The chambers on both sides ofthe aperture were then filled with 65 μl of buffer composed of 1.0 MKC1, 5 mM HEPES/KOH at pH 7.5. Silver chloride electrodes using standardmethods were placed directly into each bath and were attached to anAxopatch 200B amplifier. To paint a bilayer, a singleone-centimeter-long bristle on a 000 brush was dipped into a 3 mg per mLdiphytanoyl PC solution in spectroscopy grade hexadecane. The bristlewas then gently brushed across the aperture as viewed by a standarddissecting microscope. A 5 mV, 60 cycle square wave was applied acrossthe aperture as a seal test. Once a seal was achieved the aperture wasbrushed repeatedly with a clean bristle until a capacitance of about 0.6μF cm⁻² was achieved.

B. Insertion of individual α-hemolysin channels into theDiphylanoylPC/hexadecane bilayer.

α-hemolysin lyophilized in phosphate buffer (Calbiochem, LaJolla,Calif.) was dissolved in nanopure water at 2 μg per μl and dispensed as2 μl aliquots into 0.2 μmL polypropylene tubes. These aliquots werefrozen at −20° C. On the day of an experiment, a single tube of toxinwas placed on ice and diluted in 1.0 M KC1/HEPES buffer to a finalconcentration of 0.04 μg per μl. One μl of this diluted stock was addedto the cis side of the bilayer and mixed gently. Voltage (120 mV tranmspositive) was then applied across the bilayer. A single channeltypically inserted into the bilayer within 10-60 minutes as indicated byan abrupt increase in current. This long incubation period at low toxinconcentration (as opposed to short incubation at high concentration) waspreferable because it reduced the frequency of insertion of additionalundesired channels during experiments. In the event that no channelinsertion was observed in one hour, a second 0.04 μg aliquot of toxinwas added. This generally resulted in a channel within an additional 15minutes. Upon channel insertion, the cis chamber was immediatelyperfused with 2 mL of buffer, i.e. About 30 times the bath volume. Thesingle channels intercalated on this first attempt were of two generaltypes: i) a rectifying channel with 116-126 pA current 120 mV transpositive) vs 86 pA current 120 mV trans negative); and ii) anon-rectifying channel with approximately 50 pA current at +/−120 mV.Single rectifying channels were used immediately for nucleic acidanalysis. The low amplitude, non-rectifying channels do not translocatenucleic acids (data not shown) and were therefore removed by rupturingthe bilayer with a brief 1.3 V DC pulse. The bilayer was then reformedby passing a single bristle across the Teflon aperture using residualdiphytanolPC/hexadecane adhering to the Teflon surface. Occasionally,during bilayer reformation, a single rectifying channel of the preferredorientation would insert. If not, the bilayer was ruptured again. Thiscycle was repeated up to ten times. If no useable channel inserted afterten attempts, toxin 1 μl of the 0.04 μg per μl stock) was re-added andincubated for up to one hour as above.

The resultant bilayers are very high resistance (>200 gigaohm), lownoise 0.6 pA RMS at 5 kHz bandwidth in whole cell mode, 0.2 pA RMS inpatch mode using an Axopatch 200B amplifier), and are stable for manyhours at applied voltages in excess of 150 mV. The devices had the addedadvantages of low capacitance, small bath volume which permits use ofmicrogram quantities of nucleic acids, and facile observation of thebilayer formation process by conventional light or fluorescencemicroscopy.

II. Use of the Device

The device described above was used to characterize polymeric moleculesas follows:

A. Preparation of Polymeric Molecules

1. Preparation of RNA homopolymers.

Homopolymers of polycytidylic acid and polyadenylic acid 2000+nt) werepurchased from Fluka (Ronkonkoma, N.Y.). To generate shorter fragments,stock solutions were hydrolyzed in alkaline buffer by a modification ofan earlier technique Briefly, 5 mg of full length RNA homopolymers wereweighed into a 12 mL polypropylene tube. To this was added 1 ml ofalkaline buffer (pH 10.2, 40 mM NaHCO₃, 60 mM Na₂CO₃) pre-warrned to 60°C. For a product ranging in size from 100 to 500 nt in length, thesolution was incubated at 60° C. for 23.5 minutes and the reactionstopped by adding 100 μl of 3 M sodium acetate, pH 5.2, and 50 μl of 10%glacial acetic acid. The RNA was precipitated in 2.5 volumes of ethanolat −20° C. The pellet was rinsed in 80% ethanol, then redissolved in 1volume water and 1 volume 2×formamide loading buffer 90% formamide, 10%10× MOPS RNA buffer). The product was loaded on an 8%polyacrylamide/MOPS gel and run at 4 volts per cm alongside RNA markers(Century Markers, Ambion Inc., Austin, Tex.). The gel was then examinedby UV shadowing and RNA fragments of varying length were excised andeluted from the gel by electrophoresis. The sized RNA was thenethanol-precipitated and redissolved in water or pH 7.5 TE buffer at2-to-5 μg per μl.

2. Synthesis of DNA template for synthesis of A(30)C(70)Gp RNA.

A 134 base DNA oligo-nucleotide composed of the sequenceTAATACGACTCACTATAGGGA(A₂₉)/C(₇₀)GGTACCACACAC (SEQ ID NO:01)

was purchased from Midland Certified Reagents (Midland. Tex.).Full-length 134 nt strands were separated from incomplete strands byelectrophoresis on an 8% preparative PAGE/TBE gel at 100 V for 4 hours.The desired band was excised, the full length material was electroelutedfrom the gel slice, precipitated in ethanol, rinsed twice with 80%ethanol, air-dried, then dissolved in water to give a finalconcentration of 1 μg per μl.

Double-stranded template was synthesized from the purifiedsingle-stranded 134mer using Sequenase (Amersham/U.S. Biochemical,Cleveland, Ohio). Briefly, 1 μg of the 134mer (25 pmol final) werecombined with 0.2 μg of a 14 base reverse complement to the 3′ end ofthe 134mer 50 pmol final), 4 μl of Sequenase 5× buffer, and 3 μlnanopure water. This mixture was heated to 65° C. for 2 minutes andgradually cooled to 4° C. over 30 minutes to permit annealing of thereverse complement to the 134 nt strand. This solution was then heatedto 37° C. for two minutes and combined with 1 μl 0.1 M DTT, 2.4 μl of a2.5 mM dNTP mixture at room temperature, and 6 μl of pure water. Thissolution was brought to 37° C. for 1 minute, combined with 1 μl of 13Uper μl Sequenase and then incubated at 37° C. for 45 minutes. Theresulting double-stranded DNA product was stored at −20° C.

3. In Vitro Synthesis of A(3 0)C(70)Gp RNA.

RNA was synthesized using the 134 nt double-stranded DNA template and aT7 RNA polymerase-based kit designed to give very high yields of shorttranscripts (Megashortscript, Ambion Inc., Austin, Tex.). Briefly, wecombined, in order, at room temperature, 4 μl nanopure water, 2 μl 10×transcription buffer, 2 μl each of 75 mM ATP, CTP, UTP, GTP, 4 μl ofdsDNA template from the previous step, and 2 μl Megashortscript(T7 RNApolymerase) enzyme stock. This mixture was incubated at 37° C. for 2hours. At the end of the incubation, 1 μl of 2U/μl DNAse 1 was addedalong with 0.25 μg of RNAse T1 (Life Technologies) to cleave undesiredends of the RNA product at G residues. This digestion was incubated at37° C. for 15 minutes. The product was then run on an 8% PAGE gel in 1×MOPS RNA buffer at 80 V. The desired 101 nt band was excised and elutedby electrophoresis. The elution buffer was then exchanged for pH 7.5 TEbuffer using a Bio-Rad 30 spin column (Hercules, Calif.). The finalproduct was stored at 2 μg per μl in a −20° C. freezer.

B. Current Blockades Produced by Polymeric Molecules

1. Single Channel Clurrent Recordings.

Current readings across single α-hemolysin channels were acquired usingan Axopatch 200B integrating patch clamp amplifier (Axon Instruments,Foster City, Calif.) in voltage clamp mode. Unless otherwise noted, datawere acquired at 10 μs intervals in the whole cell configuration andwere filtered at 10 kHz using a low-pass bessel filter. The analogsignal was digitized using an Axon Instruments Digidata 1200 SeriesInterface, and then stored using Pclamp 6.02 software (Axon Instruments,Foster City, Calif.). Before the addition of RNA to the cis chamber,data were acquired in gap free format for 15 seconds each at 0, +120 mV,and −120 mV. RNA (10-15 μg) was added to the cis chamber, and blockadesof current were examined at 120 mV (trans positive) for five minutes.Blockades were stored in pClamp 6.02 (Fetchex) using the event drivenformat for five minutes.

Blockades of ionic current caused by occupancy of the α-hemolysin poreby polyadenylic acid (polyA RNA (200 μg/ml of 150±50 nt long polyA RNA))were first measured. The channel had an open current of 117 pA in 1 MKCl buffer at 120 mV potential. The polyA blockades fell into threepopulations: i) relatively short (<200 μs) blockades that reduced thecurrent by 40-60 pA; ii) blockades of indeterminate length that reducedthe channel current by 65 pA to a residual current of about 50 pA (55%blockades); and iii) blockades of 1.5 to 2.5 ms that reduced the channelcurrent by about 98 pA to 19 pA of residual current 84% blockades). Theduration of the third class of blockades was strand-length dependent,whereas the duration of the first two classes was length independent.Occasionally, polyA blockades would have a biphasic signature in whichan initial 55% blockade would transition to an 84% blockade.

The pattern of blockades caused by polyC was easily distinguishable fromthe pattern for polyA whether the polymers were examined separately orin combination. That is, the channel current was reduced significantlymore by polyC RNA 200 μg/ml of 125±/50 nucleotide long polyC) (typically95% blockades) than by polyA RNA 84% blockades) and the polyC blockadeswere shorter in duration, averaging 6 μs per nucleotide compared to 16μs per nucleotide for polyA. Also, polyC RNA rarely induced loweramplitude blockades or biphasic blockades that were very common withpolyA RNA.

This suggested that a transition from polyA to polyC segments withinindividual RNA molecules should be detectable by the α-hemolysin pore.To test this prediction, in vitro transcription and ribonuclease T1digestion was used to generate a 101-nucleotide-long RNA of the nominalcomposition A(30)C(70)Gp. Typical biphasic blockades caused by occupancyof the channel by this RNA are shown in FIG. 2. As predicted, onecomponent of the blockade reduced the channel current by 95% (consistentwith polyC RNA), and the other component reduced the current by 84 %(consistent with polyA RNA). Monophasic blockades of each characteristicamplitude were also abundant (solid arrows in FIG. 2), as were permanentblockades that required reversal of the membrane potential to be cleared(dashed arrow, FIG. 2).

That the biphasic signatures in FIG. 2 were due to the C-to-A transitionin A(30)C(70)Gp and that they could not be due to the polyA segmentalone was established as follows. Blockades caused by A(30)C(70)Gp weremeasured before and after addition of ribonuclcase A. Ribonuclease Acleaves single-stranded RNA on the 3′ end of pyrimidine residues (Davis,L. G., W. M. Kuehl, J. F. Battey. 1994. Basic Methods in MolecularBiology, 2nd Edition. Appleton and Lange, Norwalk, Conn.) and would,therefore, rapidly convert A(30)C(70)Gp to a mixture of A(30)Cp withvery short polyC oligomers and CMP. FIG. 3a shows results from thisexperiment, confirming an abrupt decrease in the frequency of biphasic95%-to-84% blockades in the presence of ribonuclease A. Thus, thebiphasic signatures shown are caused by translocation of the intactA(30)C(70)Gp strand and they cannot be accounted for by anomalousamplitude transitions in polyA RNA alone. This experiment also revealedthat the frequency of monophasic 95% blockades was significantly reduced(FIG. 3b), while the frequency of 84% blockades approximately doubledover the twenty minute incubation period (FIG. 3c), consistent withgeneration of an RNA population dominated by polyA strands.

The results demonstrate that segments of polyA and polyC in individualRNA molecules can be read during translocation of single moleculesthrough a nanometer-scale pore using the device according to the subjectinvention. This conclusion is based on the following evidence: i) RNAhomopolymers of polyA and polyC cause measurably different blockades ofcurrent in the α-hemolysin channel. Importantly, this differenceincludes the largest amplitude blockades (84% blockade for polyA vs 95%blockade for polyC) whose duration is strand-length dependent—arequirement of vectorial transport. ii) When segments of polyA and polyCare linked together in individual RNA molecules, biphasic blockades areobserved that transition from a 95% current reduction to an 84% currentreduction (FIG. 2) in a manner that is quantitatively consistent withthe homopolymer experiments. The frequency of this biphasic signature isgreatly reduced in the expected manner following cleavage ofA(30)C(70)Gp by ribonuclease A (FIG. 3).

The above results show that the subject devices can be used todistinguish between short segments of polycytidylic acid andpolyadenylic acid within individual RNA strands.

It is evident from the above results and discussion that improvedsingle-channel thin film devices are provided by the subject invention.One novel feature of the device is the conical bore, which can be moldedin a partition composed of inexpensive heat shrinkable material, such asTeflon tubing. This aspect of the invention allows the device to beproduced in large numbers as a disposable item for manufacture. Inaddition, the conical angle of the hole contributes to the stability ofthe film in the device. A second novel feature is the small size andinsulating properties of the support which lead to extremely lowelectrical noise in the output signal. The small size also allows verysmall volumes of solution to be used during the measurement. Thus, thesubject devices combine the advantages of a conical aperture with theadvantages of horizontal bilayers to yield devices having highresistance, low noise and high stability. In addition, the subjectdevices have the advantages of low capacitance and can be used toanalyze microgram quantities of nucleic acid. A third novel feature isthe U-tube electrical means which allows the user to produce ahorizontal film accessible to microscopic examination. As such, thebilayer formation process is readily observable in the subject devicesusing conventional light or fluorescence microscopy.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1 1 132 DNA Artificial Sequence synthetic polynucleotide 1 taatacgactcactataggg aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa cccccccccc 60 cccccccccccccccccccc cccccccccc cccccccccc cccccccccc cccccccccc 120 ggtaccacac ac132

What is claimed is:
 1. A single-channel thin film device comprising: acis chamber; a trans chamber; an electrical communication means forholding a conductor of electrical current connecting said cis and transchambers and having a cis terminus and a trans terminus; and a thin-filmcomprising a single channel, said thin-film covering an aperture at saidcis terminus.
 2. The device according to claim 1, wherein saidsingle-channel thin-film covered aperture comprises: a conical aperture;and wherein said single channel is a single nanopore.
 3. The deviceaccording to claim 2, wherein said nanopore is selected from the groupconsisting of a naturally occurring proteinaceous channel and asynthetic pore.
 4. The device according to claim 1, wherein said devicefurther comprises a means for applying an electric field between saidcis and trans chambers.
 5. The device according to claim 1, wherein saidelectrical communication means is a U-shaped tube connecting said cischamber with said trans chamber.
 6. In a method of monitoring a currentthrough a nanopore, the improvement comprising the steps of: applying anelectrical field between the cis and trans chambers of a single-channelthin film device according to claim 1; and monitoring said current,measuring the ionic current through said nanopore.
 7. The methodaccording to claim 6, wherein said method is a method of characterizinga naturally occurring ion channel.
 8. The method according to claim 6,wherein said method is a method of characterizing a polymeric compound.9. The method according to claim 8, wherein said method ofcharacterizing is a method of sequencing a nucleic acid.
 10. The deviceof claim 1, wherein said conductor is an electrolyte solution.
 11. Asingle-channel thin film device comprising: a cis chamber; a transchamber; a U-shaped tube connecting said cis chamber with said transchamber and having a cis terminus and a trans terminus; a conicalaperture at said cis terminus, wherein said aperture is covered with athin film comprising a single nanopore; and a means for applying anelectric field between said cis and trans chambers.
 12. The deviceaccording to claim 11, wherein said conical aperture has an innerdiameter ranging from about 1 to 50 μm.
 13. The device according toclaim 11, wherein said nanopore has an inner diameter ranging from about1 to 10 nm.
 14. The device according to claim 13, wherein said nanoporeis selected from the group consisting of a naturally occurring proteinchannel and a synthetic pore.
 15. The device according to claim 14,wherein said naturally occurring protein channel is a heptameric channelof α-hemolysin.
 16. The device according to claim 15, wherein saidchannel is a rectifying channel.
 17. A method of monitoring ioniccurrent through a nanopore, said method comprising: applying anelectrical field between the cis and trans chambers of a deviceaccording to claim 11; and measuring the ionic current through saidnanopore.
 18. A single-channel thin film device comprising: a cischamber; a trans chamber; a U-shaped tube connecting said cis chamberwith said trans chamber and having a cis terminus and a trans terminus;a conical aperture at said cis terminus, wherein said conical aperturehas an inner diameter ranging from about 1 to 50 μm and is covered witha lipid bilayer comprising a single rectifying heptameric channel ofα-hemolysin; and a means for applying an electric field between said cisand trans chambers.
 19. The device according to claim 18, wherein saidconical aperture is fabricated from Teflon.
 20. The device according toclaim 18, wherein said means for applying an electric field comprises acis electrode and a trans electrode.